Bioactive peptides and peptide derivatives of parathyroid hormone (PTH) and parathyroid hormone-related peptide (PTHrP)

ABSTRACT

Novel parathyroid hormone peptide (PTH) and parathyroid hormone related peptide (PTHrP) or derivatives thereof which are biologically active are disclosed, as are pharmaceutical compositions containing said peptides, and synthetic and recombinant methods for producing said peptides. Also disclosed are methods for treating mammalian conditions characterized by decreases in bone mass using therapeutically effective pharmaceutical compositions containing said peptides. Also disclosed are methods for screening candidate compounds of the invention for antagonistic or agonistic effects on parathyroid hormone receptor action. Also disclosed are diagnostic and therapeutic methods of said compounds.

[0001] This application claims the benefit of the filing date ofprovisional application No. 60/105,530 filed on Oct. 22, 1998, which isherein incorporated by reference.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY-SPONSOREDRESEARCH AND DEVELOPMENT

[0002] Part of the work performed during development of this inventionutilized U.S. Government funds. The U.S. Government has certain rightsin this invention.

BACKGROUND OF THE INVENTION

[0003] 1. Field of the Invention

[0004] The present invention relates to novel parathyroid hormonepeptide (PTH) derivatives and to novel parathyroid hormone-relatedpeptide (PTHrP) derivatives. In particular, the invention relates to PTHand PTHrP minimized peptide and derivatives thereof that still retainbiological activity.

[0005] 2. Description of Related Art

[0006] Parathyroid hormone (PTH) is a major regulator of calciumhomeostasis whose principal target cells occur in bone and kidney.Regulation of calcium concentration is necessary for the normal functionof the gastrointestinal, skeletal, neurologic, neuromuscular, andcardiovascular systems. PTH synthesis and release are controlledprincipally by the serum calcium level; a low level stimulates and ahigh level suppresses both hormone synthesis and release. PTH, in turn,maintains the serum calcium level by directly or indirectly promotingcalcium entry into the blood at three sites of calcium exchange: gut,bone, and kidney. PTH contributes to net gastrointestinal absorption ofcalcium by favoring the renal synthesis of the active form of vitamin D.PTH promotes calcium resorption from bone indirectly by stimulatingdifferentiation of the bone-resorbing cells, osteoclasts. It alsomediates at least three main effects on the kidney: stimulation oftubular calcium reabsorption, enhancement of phosphate clearance, andpromotion of an increase in the enzyme that completes synthesis of theactive form of vitamin D. PTH exerts these effects primarily throughreceptor-mediated activation of adenylate cyclase and phospholipase C.

[0007] Disruption of calcium homeostasis may produce many clinicaldisorders (e.g., severe bone disease, anemia, renal impairment, ulcers,myopathy, and neuropathy) and usually results from conditions thatproduce an alteration in the level of parathyroid hormone. Hypercalcemiais a condition that is characterized by an elevation in the serumcalcium level. It is often associated with primary hyperparathyroidismin which an excess of PTH production occurs as a result of a lesion(e.g., adenoma, hyperplasia, or carcinoma) of the parathyroid glands.Another type of hypercalcemia, humoral hypercalcemia of malignancy (HHM)is the most common paraneoplastic syndrome. It appears to result in mostinstances from the production by tumors (e.g., squamous, renal, ovarian,or bladder carcinomas) of a class of protein hormone which shares aminoacid homology with PTH. These PTH-related proteins (PTHrP) appear tomimic certain of the renal and skeletal actions of PTH and are believedto interact with the PTH receptor in these tissues. PTHrP is normallyfound at low levels in many tissues, including keratinocytes, brain,pituitary, parathyroid, adrenal cortex, medulla, fetal liver,osteoblast-like cells, and lactating mammary tissues. In many HHMmalignancies, PTHrP is found in the circulatory system at high levels,thereby producing the elevated calcium levels associated with HHM.

[0008] The pharmacological profiles of PTH and PTHrP are nearlyidentical in most in vitro assay systems, and elevated blood levels ofPTH (i.e., primary hyperparathyroidism) or PTHrP (i.e., HHM) havecomparable effects on mineral ion homeostasis (Broadus, A. E. & Stewart,A. F., “Parathyroid hormone-related protein: Structure, processing andphysiological actions,” in Basic and Clinical Concepts, Bilzikian, J. P.et al., eds., Raven Press, New York (1994), pp.259-294; Kronenberg, H.M. et al., “Parathyroid hormone: Biosynthesis, secretion, chemistry andaction,” in Handbook of Experimental Pharmacology, Mundy, G. R. &Martin, T. J., eds., Springer-Verlag, Heidelberg (1993), pp. 185-201).The similarities in the biological activities of the two ligands can beexplained by their interaction with a common receptor, the PTH/PTHrPreceptor, which is expressed abundantly in bone and kidney (Urena, P. etal., Endocrinology 134:451-456 (1994)).

[0009] Native human parathyroid hormone is an unmodified polypeptide of84 amino acids. It is secreted from the parathyroid glands in responseto low blood calcium levels and acts on osteoblast (bone-building cells)in bone, and on tubular epithelial cells of kidney. The hormoneinteracts with a cell surface receptor molecule, called the PTH-1receptor or PTH/PTHrP receptor, which is expressed by both osteoblastand renal tubular cells. PTHrP, the major cause of the humoralhypercalcemia of malignancy, also has normal functions that includeroles in development. PTHrP has 141 amino acids, though variants alsooccur that result from alternative gene splicing mechanisms. PTHrP playsa key role in the formation of the skeleton through a process that alsoinvolves binding to the PTH-1 receptor (Karaplis, A. C., et al., Genesand Dev. 8:277-289 (1994) and Lanske, B., et al., Science 273:663-666(1996)).

[0010] The PTH-1 receptor is homologous in primary structure to a numberof other receptors that bind peptide hormones, such as secretin(Ishihara, T. et al., EMBO J. 10:1635-1641 (1991)), calcitonin (Lin, H.Y. et al., Science 254:1022-1024 (1991)) and glucagon (Jelinek, L. J. etal., Science 259:1614-1616 (1993)); together these receptors form adistinct family called receptor family B (Kolakowski, L. F., Receptorsand Channels 2:1-7 (1994)). Within this family, the PTH-1 receptor isunique, in that it binds two peptide ligands and thereby regulates twoseparate biological processes. A recently identified PTH receptorsubtype, called the PTH-2 receptor, binds PTH but not PTHrP (Usdin, T.,et al., J. Biol. Chem. 270:15455-15458 (1995)). This observation impliedthat structural differences in the PTH and PTHrP ligands determinedselectivity for interaction with the PTH-2 receptor. The PTH-2 receptorhas been detected by RNA methods in the brain, pancreas and vasculature,however, its biological function has not been determined (Usdin, T., etal., J. Biol. Chem. 270:15455-15458 (1995)). It is hypothesized that thefamily B receptors use a common molecular mechanism to engage their owncognate peptide hormone (Bergwitz, C., et al., J. Biol. Chem.271:26469-26472 (1996)).

[0011] The binding of either radiolabeled PTH(1-34) or PTHrP(1-36) tothe PTH-1 receptor is competitively inhibited by either unlabeled ligand(Jüppner, H. et al., J. Biol. Chem. 263:8557-8560 (1988); Nissenson, R.A. et al., J. Biol. Chem. 263:12866-12871 (1988)). Thus, the recognitionsites for the two ligands in the PTH-1 receptor probably overlap. Inboth PTH and PTHrP, the 15-34 region contains the principal determinantsfor binding to the PTH-1 receptor. Although these regions show onlyminimal sequence homology (only 3 amino acid identities), each 15-34peptide can block the binding of either PTH(1-34) or PTHrP(1-34) to thePTH-1 receptor (Nussbaum, S. R. et al., J. Biol. Chem. 255:10183-10187(1980); Caulfield, M. P. et al., Endocrinology 127:83-87 (1990);Abou-Samra, A.-B. et al., Endocrinology 125:2215-2217 (1989)). Further,the amino terminal portion of each ligand is required for bioactivity,and these probably interact with the PTH-1 receptor in similar ways,since 8 of 13 of these residues are identical in PTH and PTHrP.

[0012] Both PTH and PTHrP bind to the PTH-1 receptor with affinity inthe nM range; the ligand-occupied receptor transmits a “signal” acrossthe cell membrane to intracellular effector enzymes through a mechanismthat involves intermediary heterotrimeric GTP-binding proteins (Gproteins). The primary intracellular effector enzyme activated by thePTH-1 receptor in response to PTH or PTHrP is adenylyl cyclase (AC).Thus, PTH induces a robust increase in the “second messenger” molecule,cyclic adenosine monophosphate (cAMP) which goes on to regulate thepoorly characterized “downstream” cellular processes involved inbone-remodeling (both bone formation and bone resorption processes). Incertain cell-based assay systems, PTH can stimulate effector enzymesother than AC, including phospholipase C (PLC), which results inproduction of inositol triphosphate (IP₃), diacylglycerol (DAG) andintracellular calcium (iCa²⁺). The roles of these non-cAMP secondmessenger molecules in bone metabolism are presently unknown.

[0013] Osteoporosis is a potentially crippling skeletal disease observedin a substantial portion of the senior adult population, in pregnantwomen and even in juveniles. The disease is marked by diminished bonemass, decreased bone mineral density (BMD), decreased bone strength andan increased risk of bone fracture. At present, there is no effectivecure for osteoporosis, though estrogen, calcitonin and thebisphosphonates, etidronate and alendronate are used to treat thedisease with varying levels of success through their action to decreasebone resorption. Since parathyroid hormone regulates blood calcium andthe phosphate levels, and has potent anabolic (bone-forming) effects onthe skeleton, in animals (Shen, V., et al., Calcif. Tissue Int.50:214-220 (1992); Whitefild, J. F., et al., Calcif. Tissue Int.56:227-231 (1995) and Whitfield, J. F., et al., Calcif. Tissue Int.60:26-29 (1997)) and humans (Slovik, D. M., et al., J. Bone Miner. Res.1:377-381 (1986); Dempster, D. W., et al., Endocr. Rev. 14:690-709(1993) and Dempster, D. W., et al., Endocr. Rev. 15:261 (1994)) whenadministered intermittently, PTH, or PTH derivatives, are primecandidates for new and effective therapies for osteoporosis.

[0014] Truncated PTH derivatives such as PTH(1-34) and PTH(1-3 1) areactive in most assay systems and promote bone-formation (Whitefild, J.F., et al., Calcif. Tissue Int. 56:227-231 (1995); Whitfield, J. F., etal., Calcif. Tissue Int. 60:26-29 (1997); Slovik, D. M., et al., J. BoneMiner. Res. 1:377-381 (1986); Tregear, G. W., et al., Endocrinology93:1349-1353 (1973); Rixon, R. H., et al., J. Bone Miner. Res.9:1179-1189 (1994); Whitfield, J. F. and Morley, P., Trends Pharmacol.Sci. 16:372-386 (1995) and Whitfield, J. F., et al., Calcif. Tissue Int.58:81-87(1996)). But these peptides are still too large for efficientnon-parenteral delivery and low cost. The discovery of an even smaller“minimized” version of PTH or PTHrP would be an important advance in theeffort to develop new treatments for osteoporosis.

[0015] PTH and PTHrP derivatives that have amino acid substitutions ordeletions in the 1-14 region usually exhibit diminished activity(Tregear, G. W., et al., Endocrinology 93:1349-1353 (1973); Goltzman,D., et al., J. Biol. Chem. 250:3199-3203 (1975); Horiuchi, N., et al.,Science 220:1053-1055 (1983) and Gardella, T. J., et al., J. Biol. Chem.266:13141-13146 (1991))

[0016] Several short NH₂-terminal PTH or PTHrP peptides have beeninvestigated previously, but no activity was detected. For example,bPTH(1- 12) was inactive in adenylyl cyclase assays performed in ratrenal membranes (Rosenblatt, M., “Parathyroid Hormone: Chemistry andStructure-Activity Relations,” in Pathobiology Annual, Ioachim, H. L.,ed., Raven Press, New York (1981), pp. 53-84) and PTHrP(1-16) wasinactive in AC assays performed in Chinese hamster ovary (CHO) cellsexpressing the cloned rat PTH-1 receptor (Azurani, A., et al., J. Biol.Chem. 271:14931-14936 (1996)). It has been known that residues in the15-34 domain of PTH contribute importantly to receptor binding affinity,as the PTH(15-34) fragment binds weakly to the receptor, but thispeptide does not activate AC (Naussbaum, S. R., et al., J. Biol. Chem.255:10183-10187 (1980) and Gardella, T. J., et al., Endocrinology132:2024-2030 (1993)).

SUMMARY OF THE INVENTION

[0017] The relatively large size of native PTH or PTHrP presentschallenges to the use of these peptides as treatments for osteoporosis.In general, a protein of this size is not suitable for use as a drug,since it cannot be delivered effectively by simple methods such as nasalinhalation. Instead, injection is required, and in the case of PTH,daily, or almost daily injections would most likely be needed to achieveincreases in bone formation rates. Additionally, larger peptides aretechnically difficult and expensive to prepare by conventional syntheticchemistry methods. Alternative methods employing recombinant DNA andcell-based expression systems are also expensive, potentially vulnerableto contamination by foreign proteins and do not circumvent the deliveryproblem.

[0018] Accordingly, it would be advantageous for those skilled in theart to be able to identify a small molecule analog (either peptide ornon-peptide) that is based on the larger peptide and yet which stillretains the desired biological activities. The activity may at first beweak relative to the intact peptide, but further optimization can leadto enhanced efficacy and potency.

[0019] The present invention relates to PTH(1-14)/PTHrP(1-14) peptidesand derivatives thereof. Compounds of the invention which includePTH(1-14)/PTHrP(1-14) peptides, fragments thereof, derivatives thereof,pharmaceutically acceptable salts thereof, and N- or C-derivativesthereof, are hereinafter collectively referred to as “compounds of SEQID NO:1 and derivatives thereof”.

[0020] In detail, the invention provides synthetic and/or recombinantbiologically active peptide derivatives of PTH(1-14) and PTHrP(1-14). Inone specific embodiment, this invention provides a biologically activepeptide at least 85% identical to a peptide consisting essentially ofthe formula:

[0021] (a) X₀₁ ValSerGluX₀₂GlnLeuX₀₃HisX₀₄X₀₅GlyLysX₀₆(SEQ ID NO:1);

[0022] (b) fragments thereof containing amino acids 1-9, 1-10,1-11,1-12, or 1-13;

[0023] (c) pharmaceutically acceptable salts thereof; or

[0024] (d) N- or C-derivatives thereof,

[0025] wherein:

[0026] X₀₁ is Ser or Ala;

[0027] X₀₂ is Ile or His;

[0028] X₀₃ is Met, Leu or Nle;

[0029] X₀₄ is Asn or Asp;

[0030] X₀₅ is Leu or Lys; and

[0031] X₀₆ is His or Ser, provided that said peptide is not PTHrP(1-14).

[0032] In accordance with yet a further aspect of the invention, thisinvention also provides pharmaceutical compositions comprising

[0033] (a) a biologically active peptide at least 85% identical to apeptide consisting essentially of the formula:SerValSerGluIleGlnLeuMetHisAsnLeu GlyLysHis (SEQ ID NO:3);

[0034] (b) fragments thereof containing amino acids 1-9, 1-10, 1-11,1-12, or 1-13;

[0035] (c) pharmaceutically acceptable salts thereof; or

[0036] (d) N- or C-derivatives thereof; and a pharmaceuticallyacceptable carrier.

[0037] In accordance with yet a further aspect of the invention, thisinvention also provides pharmaceutical compositions comprising

[0038] (a) a biologically active peptide at least 85% identical to apeptide consisting essentially of the formula:AlaValSerGluHisGlnLeuLeuHisAspLys GlyLysSer (SEQ ID NO:4);

[0039] (b) fragments thereof containing amino acids 1-9, 1-10, 1-11,1-12, or 1-13;

[0040] (c) pharmaceutically acceptable salts thereof; or

[0041] (d) N- or C-derivatives thereof; and a pharmaceuticallyacceptable carrier.

[0042] In accordance with yet a further aspect of the invention, thisinvention provides a nucleic acid molecule consisting essentially of apolynucleotide encoding a biologically active peptide which has an aminoacid sequence selected from the group consisting of:

[0043] (a) X₀₁ ValSerGluX₀₂GlnLeuX₀₃HisX₀₄X₀₅GlyLysX₀₆(SEQ ID NO:1); or

[0044] (b) fragments thereof containing amino acids 1-9, 1-10, 1-11,1-12, or 1-13;

[0045] wherein:

[0046] X₀₁ is Ser or Ala;

[0047] X₀₂ is Ile or His;

[0048] X₀₃ is Met, Leu or Nle;

[0049] X₀₄ is Asn or Asp;

[0050] X₀₅ is Leu or Lys; and

[0051] X₀₆ is His or Ser,

[0052] provided that said peptide is not PTHrP(1-14).

[0053] In accordance with yet a further aspect of the invention, thisinvention provides a recombinant DNA molecule comprising: (1) anexpression control region, said region in operable linkage with (2) apolynucleotide sequence coding for a biologically active peptide,wherein said peptide is selected from the group consisting of:

[0054] (a) X₀₁ ValSerGluX₀₂GlnLeuX₀₃HisX₀₄X₀₅GlyLysX₀₆(SEQ ID NO:1); or

[0055] (b) fragments thereof containing amino acids 1-9, 1-10, 1-11,1-12, or 1-13;

[0056] wherein:

[0057] X₀₁ is Ser or Ala;

[0058] X₀₂ is Ile or His;

[0059] X₀₃ is Met, Leu or Nle;

[0060] X₀₄ is Asn or Asp;

[0061] X₀₅ is Leu or Lys; and

[0062] X₀₆ is His or Ser,

[0063] provided that said peptide is not PTHrP(1-14).

[0064] In accordance with yet a further aspect of the invention, thisinvention provides a method for treating mammalian conditionscharacterized by decreases in bone mass, which method comprisesadministering to a subject in need thereof an effective bonemass-increasing amount of a biologically active peptide, wherein saidpeptide comprises an amino acid sequence at least 85% identical to amember selected from the group consisting essentially of:

[0065] (a) X₀₁ ValSerGluX₀₂GlnLeuX₀₃HisX₀₄X₀₅GlyLysX₀₆(SEQ ID NO:1);

[0066] (b) fragments thereof containing amino acids 1-9, 1-10, 1-11,1-12, or 1-13;

[0067] (c) pharmaceutically acceptable salts thereof; or

[0068] (d) N- or C-derivatives thereof;

[0069] wherein:

[0070] X₀₁ is Ser or Ala;

[0071] X₀₂ is Ile or His;

[0072] X₀₃ is Met, Leu or Nle;

[0073] X₀₄ is Asn or Asp;

[0074] X₀₅ is Leu or Lys; and

[0075] X₀₆ is His or Ser,

[0076] provided that said peptide is not PTHrP(1-14); and apharmaceutically acceptable carrier.

[0077] In accordance with yet a further aspect of the invention, thisinvention provides a method for the treatment of a patient having needof a biologically active peptide comprising administering atherapeutically effective amount of a peptide, wherein said peptidecomprises an amino acid sequence at least 85% identical to a memberselected from the group consisting essentially of:

[0078] (a) a biologically active peptide consisting essentially of theformula: SerValSerGluIleGlnLeuMetHisAsnLeuGlyLysHis (SEQ ID NO:3);

[0079] (b) fragments thereof containing amino acids 1-9, 1-10, 1-11,1-12, or 1-13;

[0080] (c) N- or C-derivatives thereof; or

[0081] (d) pharmaceutically acceptable salts thereof; and apharmaceutically acceptable carrier.

[0082] In accordance with yet a further aspect of the invention, thisinvention provides a method for the treatment of a patient having needof a biologically active peptide comprising administering atherapeutically effective amount of a peptide, wherein said peptidecomprises an amino acid sequence at least 85% identical to a memberselected from the group consisting essentially of:

[0083] (a) a biologically active peptide consisting essentially of theformula: AlaValSerGluHisGlnLeuLeuHisAspLysGlyLysSer (SEQ ID NO:4);

[0084] (b) fragments thereof containing amino acids 1-9, 1-10, 1-11,1-12, or 1-13;

[0085] (c) N- or C-derivatives thereof; or

[0086] (d) pharmaceutically acceptable salts thereof; and apharmaceutically acceptable carrier.

[0087] In accordance with yet a further aspect of the invention, thereis provided a method for treating a medical disorder that results fromaltered or excessive action of the PTH-1/PTH-2 receptor, comprisingadministering to a patient a therapeutically effective amount of abiologically active peptide wherein said peptide comprises an amino acidsequence at least 85% identical to a member selected from the groupconsisting essentially of:

[0088] (a) X₀₁ ValSerGluX₀₂GlnLeuX₀₃HisX₀₄X₀₅GlyLysX₀₆(SEQ ID NO: 1);

[0089] (b) fragments thereof containing amino acids 1-9, 1-10, 1-11,1-12, or 1-13;

[0090] (c) pharmaceutically acceptable salts thereof; or

[0091] (d) N- or C-derivatives thereof;

[0092] wherein:

[0093] X₀₁ is Ser or Ala;

[0094] X₀₂ is Ile or His;

[0095] X₀₃ is Met, Leu or Nle;

[0096] X₀₄ is Asn or Asp;

[0097] X₀₅ is Leu or Lys; and

[0098] X₀₆ is His or Ser,

[0099] provided said peptide is not PTHrP(1-14); and a pharmaceuticallyacceptable carrier sufficient to inhibit activation of the PTH-1/PTH-2receptor of said patient.

[0100] In accordance with yet a further aspect of the invention, thisinvention also provides a method for determining rates of bonereformation, bone resorption and/or bone remodeling comprisingadministering to a patient an effective amount of a labeled peptide ofSEQ ID NO: 1 or a derivative thereof and determining the uptake of saidpeptide into the bone of said patient. The peptide may be labeled with alabel selected from the group consisting of: radiolabel, flourescentlabel, bioluminescent label, or chemiluminescent label. An example of asuitable radiolabel is ^(99m)Tc.

[0101] In accordance with yet a further aspect of the invention, anyamino-acid substitutions at positions 1-9, and more particularly thoseamino acid substitutions at amino acid positions 10, 11, 12, 13, and/or14, which do not destroy the biological activity of thePTH(1-14)/PTHrP(1-14) peptide analog to antagonize or agonize thePTH-1/PTH-2 receptor (as determined by assays known to the skilledartisan and discussed below), are also included within the scope of thepresent invention.

BRIEF DESCRIPTION OF THE FIGURES

[0102]FIG. 1. Bioactivity of amino-terminal and carboxy-terminalfragments of PTH(1-34). Fragments of parathyroid hormone weresynthesized by chemical methods and purified by reverse-phase HPLC.Peptides were tested for the ability to stimulate cAMP accumulation inCOS-7 cells expressing the cloned human PTH-1 receptor. PTH(1-34) wastested at a dose of 1 μM, other peptides were tested at 67 μM. Peptideswere tested in duplicate (±s.e.m.) at a dose of 67 μM. The cAMP incontrol untreated cells is indicated by Bsl. Cells were treated for 30minutes at 21° C.

[0103]FIG. 2. Alanine-scan of PTH(1-14). Shown are the bioactivities of14 different PTH(1-14) derivatives, each having a different amino acidof the native sequence (shown at bottom of figure) replaced by alanine.Peptides were chemically synthesized, purified and tested for ability tostimulate cAMP formation in COS-7 cells expressing the cloned humanPTH-1 receptor. Peptides were tested in duplicate (±s.e.m.) at a dose of67 μM. As a control, untreated cells, indicated by basal, were measured.The PTH(1-14) containing alanine at position 1 was used as the wild-typereference. Cells were stimulated for 30 minutes at 21° C.

[0104]FIG. 3. cAMP dose response curves of short amino-terminal PTHanalogs in LLC-PK1 cells stably transfected with the human PTH-1receptor (LLC-B7 cells). LLC-B7 cells in 24 plates were treated with theindicated peptides for 60 mins at 21° C., and then intracellular cAMPlevels were measured. All PTH peptides shown are based on the rat PTHsequence and are carboxy-terminally amidated. As can be seen, there isno gain in activity when the PTH(1-14) peptide is extended to residue15; and PTH(1-13) or shorter analogs exhibit only very weak activity.

[0105]FIG. 4. cAMP dose response curves of PTH(1-14) in LLC-B7 cells andin untransfected LLC-PK1 cells. PTH(1-14) and PTH(1-34) control peptideswere tested as in FIG. 3. As can be seen, the response to PTH(1-14) inthese cells is completely dependent on the presence of the PTH-1receptor.

[0106]FIG. 5. Alanine-scan of PTH(1-14) in LLC-B7 cells. Each PTH(1-14)was tested in duplicate at a dose of 100 μM. LLC-B7 cells in 24 plateswere treated with the indicated peptides, for 60 mins at 21° C., andthen intracellular cAMP levels were measured.

[0107]FIG. 6. Specificity of PTH(1-14). The analog PTH(1-14) was testedin COS-7 cells transfected with rat secretin receptor, which respondsfully to the control native secretin (1-27). PTH(1-14) does notstimulate cAMP in these cells. Thus, the response to PTH(1-14) isdependent on the presence of the PTH-1 receptor.

[0108]FIG. 7. cAMP activity of [Ile5]PTHrP(1-14). LLC-B7 cells weretreated with the indicated peptide ligand, each at 100 μM, and thenintracellular cAMP levels were measured.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0109] Definitions

[0110] In the description that follows, a number of terms used inrecombinant DNA technology and peptide synthesis are utilizedextensively. In order to provide a clear and consistent understanding ofthe specification and claims, including the scope to be given suchterms, the following definitions are provided.

[0111] Cloning vector: A plasmid or phage DNA or other DNA sequencewhich is able to replicate autonomously in a host cell, and which ischaracterized by one or a small number of restriction endonucleaserecognition sites at which such DNA sequences may be cut in adeterminable fashion without loss of an essential biological function ofthe vector, and into which a DNA fragment may be spliced in order tobring about its replication and cloning. The cloning vector may furthercontain a marker suitable for use in the identification of cellstransformed with the cloning vector. Markers, for example, providetetracycline resistance or ampicillin resistance.

[0112] Expression vector: A vector similar to a cloning vector but whichis capable of enhancing the expression of a gene which has been clonedinto it, after transformation into a host. The cloned gene is usuallyplaced under the control of (i. e., operably linked to) certain controlsequences such as promoter sequences. Promoter sequences may be eitherconstitutive or inducible.

[0113] Recombinant Host: According to the invention, a recombinant hostmay be any prokaryotic or eukaryotic host cell which contains thedesired cloned genes on an expression vector or cloning vector. Thisterm is also meant to include those prokaryotic or eukaryotic cells thathave been genetically engineered to contain the desired gene(s) in thechromosome or genome of that organism. For examples of such hosts, seeSambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition,Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1989).Preferred recombinant hosts are eukaryotic cells transformed with theDNA construct of the invention. More specifically, mammalian cells arepreferred.

[0114] Promoter: A DNA sequence generally described as the 5′ region ofa gene, located proximal to the start codon. The transcription of anadjacent gene(s) is initiated at the promoter region. If a promoter isan inducible promoter, then the rate of transcription increases inresponse to an inducing agent. In contrast, the rate of transcription isnot regulated by an inducing agent if the promoter is a constitutivepromoter. Examples of promoters include the CMV promoter (InVitrogen,San Diego, Calif.), the SV40, MMTV, and hMTIIa promoters (U.S. Pat. No.5,457,034), the HSV-1 4/5 promoter (U.S. Pat. No. 5,501,979), and theearly intermediate HCMV promoter (WO92/17581). Also, tissue-specificenhancer elements may be employed. Additionally, such promoters mayinclude tissue and cell-specific promoters of an organism.

[0115] Polynucleotide: This term generally refers to anypolyribonucleotide or polydeoxribonucleotide, which may be unmodifiedRNA or DNA or modified RNA or DNA. “Polynucleotides” include, withoutlimitation single- and double-stranded DNA, DNA that is a mixture ofsingle- and double-stranded regions, single- and double-stranded RNA,and RNA that is mixture of single- and double-stranded regions, hybridmolecules comprising DNA and RNA that may be single-stranded or, moretypically, double-stranded or a mixture of single- and double-strandedregions. In addition, “polynucleotide” refers to triple-stranded regionscomprising RNA or DNA or both RNA and DNA. The term polynucleotide alsoincludes DNAs or RNAs containing one or more modified bases and DNAs orRNAs with backbones modified for stability or for other reasons.“Modified” bases include, for example, tritylated bases and unusualbases such as inosine. A variety of modifications have been made to DNAand RNA; thus, “polynucleotide” embraces chemically, enzymatically ormetabolically modified forms of polynucleotides as typically found innature, as well as the chemical forms of DNA and RNA characteristic ofviruses and cells. “Polynucleotide” also embraces relatively shortpolynucleotides, often referred to as oligonucleotides.

[0116] Polypeptide: This term refers to any peptide or proteincomprising two or more amino acids joined to each other by peptide bondsor modified peptide bonds, i.e., peptide isosteres. “Polypeptide” refersto both short chains, commonly referred to as peptides, oligopeptides oroligomers, and to longer chains, generally referred to as proteins.Polypeptides may contain amino acids other than the 20 gene-encodedamino acids. “Polypeptides” include amino acid sequences modified eitherby natural processes, such as post-translational processing, or bychemical modification techniques which are well known in the art. Suchmodifications are well described in basic texts and in more detailedmonographs, as well as in the research literature. Modifications canoccur anywhere in a polypeptide, including the peptide backbone, theamino acid side-chains and the amino or carboxyl termini. It will beappreciated that the same type of modification may be present in thesame or varying degrees at several sites in a given polypeptide. Also, agiven polypeptide may contain many types of modifications.

[0117] Polypeptides may be branched and they may be cyclic, with orwithout branching. Cyclic, branched and branched cyclic polypeptides mayresult from post-translation natural processes or may be made bysynthetic methods. Modifications include acetylation, acylation,ADP-ribosylation, amidation, covalent attachment of flavin, covalentattachment of a heme moiety, covalent attachment of a nucleotide ornucleotide derivative, covalent attachment of a lipid or lipidderivative, covalent attachment of phosphotidylinositol, cross-linking,cyclization, disulfide bond formation, demethylation, formation ofcovalent cross-links, formation of cystine, formation of pyroglutamate,formylation, gamma-carboxylation, glycosylation, GPI anchor formation,hydroxylation, iodination, methylation, myristoylation, oxidation,proteolytic processing, phosphorylation, prenylation, racemization,selenoylation, sulfation, transfer-RNA mediated addition of amino acidsto proteins such as arginylation, and ubiquitination. See, for instance,Proteins-Structure and Molecular Properties, 2nd Ed., T. E. Creighton,W. H. Freeman and Company, New York, 1993 and Wold, F.,Posttranslational Protein Modifications: Perspectives and Prospects,pgs. 1-12 in Posttranslational Covalent Modification of Proteins, B. C.Johnson, Ed., Academic Press, New York, 1983; Seifter et al., “Analysisfor protein modifications and nonprotein cofactors”, Methods in Enzymol.182:626-646 (1990) and Rattan et al., “Protein Synthesis:Posttranslational Modifications and Aging”, Ann NY Acad Sci 663:48-62(1992).

[0118] Homologous/Nonhomologous: Two nucleic acid molecules areconsidered to be “homologous” if their nucleotide sequences share asimilarity of greater than 40%, as determined by HASH-coding algorithms(Wilber, W. J. and Lipman, D. J., Proc. Natl. Acad. Sci. 80:726-730(1983)). Two nucleic acid molecules are considered to be “nonhomologous”if their nucleotide sequences share a similarity of less than 40%.

[0119] Isolated: A term meaning altered “by the hand of man” from thenatural state. If an “isolated” composition or substance occurs innature, it has been changed or removed from its original environment, orboth. For example, a polynucleotide or a polypeptide naturally presentin a living animal is not “isolated,” but the same polynucleotide orpolypeptide separated from the coexisting materials of its natural stateis “isolated”, as the term is employed herein. Thus, a polypeptide orpolynucleotide produced and/or contained within a recombinant host cellis considered isolated for purposes of the present invention. Alsointended as an “isolated polypeptide” or an “isolated polynucleotide”are polypeptides or polynucleotides that have been purified, partiallyor substantially, from a recombinant host cell or from a native source.For example, a recombinantly produced version of compounds of SEQ IDNO:1 and derivatives thereof can be substantially purified by theone-step method described in Smith and Johnson, Gene 67:31-40 (1988).

[0120] By “isolated” is meant that the DNA is free of the codingsequences of those genes that, in the naturally-occurring genome of theorganism (if any) from which the DNA of the invention is derived,immediately flank the gene encoding the DNA of the invention. Theisolated DNA may be single-stranded or double-stranded, and may begenomic DNA, cDNA, recombinant hybrid DNA, or synthetic DNA. It may beidentical to a native DNA sequence encoding compounds of SEQ ID NO:1 andderivatives thereof, or may differ from such sequence by the deletion,addition, or substitution of one or more nucleotides. Single-strandedDNAs of the invention are generally at least 8 nucleotides long,(preferably at least 18 nucleotides long, and more preferably at least30 nucleotides long) ranging up to full length of the DNA moleculeencoding compounds of SEQ ID NO:1 and derivatives thereof (i.e., 42nucleotides); they preferably are detectably labeled for use ashybridization probes, and may be antisense.

[0121] High Stringency: By “high stringency” is meant, for example,conditions such as those described for the isolation of cDNA (also seeCurrent Protocols in Molecular Biology, John Wiley & Sons, New York(1989), hereby incorporated by reference). The DNA of the invention maybe incorporated into a vector [which may be provided as a purifiedpreparation (e.g., a vector separated from the mixture of vectors whichmake up a library)] containing a DNA sequence encoding a peptide of theinvention (e.g. compounds of SEQ ID NO:1 and derivatives thereof) and acell or essentially homogenous population of cells (e.g., prokaryoticcells, or eukaryotic cells such as mammalian cells) which contain thevector (or the isolated DNA described above).

[0122] Identity: This term refers to a measure of the identity ofnucleotide sequences or amino acid sequences. In general, the sequencesare aligned so that the highest order match is obtained. “Identity” perse has an art-recognized meaning and can be calculated using publishedtechniques. (See, e.g.: Computational Molecular Biology, Lesk, A. M.,ed., Oxford University Press, New York, 1988; Biocomputing: Informaticsand Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993;Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin,H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis inMolecular Biology, von Heinje, G., Academic Press, 1987; and SequenceAnalysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press,New York, 1991). While there exist a number of methods to measureidentity between two polynucleotide or polypeptide sequences, the term“identity” is well known to skilled artisans (Carillo, H. & Lipton, D.,SIAM J Applied Math 48:1073 (1988)). Methods commonly employed todetermine identity or similarity between two sequences include, but arenot limited to, those disclosed in Guide to Huge Computers, Martin J.Bishop, ed., Academic Press, San Diego, 1994, and Carillo, H. & Lipton,D., SIAM J Applied Math 48:1073 (1988). Methods to determine identityand similarity are codified in computer programs. Preferred computerprogram methods to determine identity and similarity between twosequences include, but are not limited to, GCG program package(Devereux, J., et al., Nucleic Acids Research 12(i):387 (1984)), BLASTP,BLASTN, FASTA (Atschul, S. F., et al., J Molec Biol 215:403 (1990)).

[0123] Therefore, as used herein, the term “identity” represents acomparison between a test and reference polypeptide. More specifically,reference test polypeptide is defined as any polypeptide that is 85% ormore identical to a reference polypeptide. As used herein, the term atleast 85% identical to refers to percent identities from 85 to 99.99relative to the reference polypeptides. Identity at a level of 85% ormore is indicative of the fact that, assuming for exemplificationpurposes a test and reference polynucleotide length of 100 amino acids,that no more than 15% (i.e., 15 out of 100) amino acids in the testpolypeptides differ from that of the reference polypeptides. Suchdifferences may be represented as point mutations randomly distributedover the entire length of the amino acid sequence of the invention orthey may be clustered in one or more locations of varying length up tothe maximum allowable 2/14 amino acid difference (approximately 85%identity). Differences are defined as amino acid substitutions, ordeletions.

[0124] Fragment: A “fragment” of a molecule such as a compound of SEQ IDNO: 1 or derivative thereof is meant to refer to any polypeptide subsetof these molecules.

[0125] Functional Derivative: The term “derivatives” is intended toinclude “variants,” the “derivatives,” or “chemical derivatives” of themolecule. A “variant” of a molecule such as a compound of SEQ ID NO: 1or derivative thereof is meant to refer to a molecule substantiallysimilar to either the entire molecule, or a fragment thereof. An“analog” of a molecule such as a compound of SEQ ID NO: 1 or derivativethereof is meant to refer to a non-natural molecule substantiallysimilar to either the SEQ ID NO: 1 molecules or fragments thereof.

[0126] A molecule is said to be “substantially similar” to anothermolecule if the sequence of amino acids in both molecules issubstantially the same, and if both molecules possess a similarbiological activity. Thus, provided that two molecules possess a similaractivity, they are considered variants, derivatives, or analogs as thatterm is used herein even if one of the molecules contains additionalamino acid residues not found in the other, or if the sequence of aminoacid residues is not identical.

[0127] As used herein, a molecule is said to be a “chemical derivative”of another molecule when it contains additional chemical moieties notnormally a part of the molecule. Such moieties may improve themolecule's solubility, absorption, biological half-life, etc. Themoieties may alternatively decrease the toxicity of the molecule,eliminate or attenuate any undesirable side effect of the molecule, etc.Examples of moieties capable of mediating such effects are disclosed inRemington's Pharmaceutical Sciences (1980) and will be apparent to thoseof ordinary skill in the art.

[0128] Biological Activity of the Protein: This expression refers to themetabolic or physiologic function of compounds of SEQ ID NO: 1 orderivatives thereof including similar activities or improved activitiesor those activities with decreased undesirable side-effects. Alsoincluded are antigenic and immunogenic activities of said compounds ofSEQ ID NO: 1 or derivatives thereof.

[0129] Fusion protein: By the term “fusion protein” is intended a fusedprotein comprising compounds of SEQ ID NO: 1 or derivatives thereof,either with or without a “selective cleavage site” linked at itsN-terminus, which is in turn linked to an additional amino acid leaderpolypeptide sequence.

[0130] Selective cleavage site: The term “selective cleavage site”refers to an amino acid residue or residues which can be selectivelycleaved with either chemicals or enzymes in a predictable manner. Aselective enzyme cleavage site is an amino acid or a peptide sequencewhich is recognized and hydrolyzed by a proteolytic enzyme. Examples ofsuch sites include, without limitation, trypsin or chymotrypsin cleavagesites.

[0131] Leader Sequence: By the term “leader sequence” is intended apolynucleotide sequence linked to compounds of SEQ ID NO: 1, andexpressed in host cells as a fusion protein fused to the selectivecleavage site and compounds of SEQ ID NO: 1. The term “leaderpolypeptide” describes the expressed form of the “leader sequence” asobtained in the fusion protein.

[0132] The fusion protein, which is often insoluble and found ininclusion bodies when it is overexpressed, is purified from otherbacterial protein by methods well known in the art. In a preferredembodiment, the insoluble fusion protein is centrifuged and washed aftercell lysis, and resolubilized with guanidine-HCl. It can remain solubleafter removal of the denaturant by dialysis. (For purification ofrefractile proteins, see Jones, U.S. Pat. No. 4,512,922; Olson, U.S.Pat. No. 4,518,526; and Builder et al., U.S. Pat. Nos. 4,511,502 and4,620,948).

[0133] The recombinantly produced compounds of SEQ ID NO: 1 orderivatives thereof can be purified to be substantially free of naturalcontaminants from the solubilized fusion protein through the use of anyof a variety of methodologies. As used herein, a compound is said to be“substantially free of natural contaminants” if it has beensubstantially purified from materials with which it is found followingexpression in bacterial or eukaryotic host cells. Compounds of SEQ IDNO: 1 or derivatives thereof may be purified through application ofstandard chromatographic separation technology.

[0134] Alternatively, the peptide may be purified using immuno-affinitychromatography (Rotman, A. et al., Biochim. Biophys. Acta 641:114-121(1981); Sairam, M. R. J,. Chromatog 215:143-152 (1981); Nielsen, L. S.et al., Biochemistry 21:6410-6415 (1982); Vockley, J. et al., Biochem.J. 217:535-542 (1984); Paucha, E. et al., J. Virol. 51:670-681 (1984);and Chong, P. et al., J. Virol. Meth. 10:261-268 (1985)).

[0135] After partial or substantial purification, the fusion protein istreated enzymatically with the enzyme corresponding to the cleavagesite. Alternatively, the fusion protein in its more impure state, evenin refractile form, can be treated with the enzyme. If needed, theresulting mature compounds of SEQ ID NO: 1 or derivatives thereof, canbe further purified. Conditions for enzymatic treatment are known tothose of skill in the art.

[0136] Gene Therapy: A means of therapy directed to altering the normalpattern of gene expression of an organism. Generally, a recombinantpolynucleotide is introduced into cells or tissues of the organism toeffect a change in gene expression.

[0137] Host Animal: Transgenic animals, all of whose germ and somaticcells contain the DNA construct of the invention. Such transgenicanimals are in general vertebrates. Preferred host animals are mammalssuch as non-human primates, mice, sheep, pigs, cattle, goats, guineapigs, rodents, e.g. rats, and the like. The term Host Animal alsoincludes animals in all stages of development, including embryonic andfetal stages.

[0138] I. Compounds of SEQ ID NO: 1 and Derivatives Thereof—Structuraland Functional Properties

[0139] We describe herein a novel “minimized” variant of PTH thatretains bioactivity, and is small enough to be deliverable by simplenon-injection methods. The new peptide corresponds to the 1-14 sequenceof native PTH or shorter variants thereof and thus has a molecularweight of less than 2,000 daltons. The present invention pertains to thenative PTH(1-14) peptide, the 1-14 sequence of PTH-related peptide(PTHrP), and peptide derivatives derived from these peptides byalteration in amino acid composition or amino acid chain length.

[0140] The primary amino acid sequence of the native PTH(1-14) peptide(N-terminus to C-terminus) is SerValSerGluIleGlnLeuMetHisAsnLeuGlyLysHis(SEQ ID NO: 3), whereas the primary amino acid sequence of the nativePTHrP(1-14) peptide (N-terminus to C-terminus) isAlaValSerGluHisGlnLeuLeu HisAspLysGlyLysSer (SEQ ID NO: 4). Accordingly,the peptide sequence in common between Sequence ID NOs: 3 and 4 consistsof the following generic formula:

X₀₁ValSerGluX₀₂GlnLeuX₀₃HisX₀₄X₀₅GlyLysX₀₆ (SEQ ID NO:1)

[0141] wherein:

[0142] X₀₁ is Ser or Ala;

[0143] X₀₂ is Ile or His;

[0144] X₀₃ is Met, Leu, or Nle;

[0145] X₀₄ is Asn or Asp;

[0146] X₀₅ is Leu or Lys; and

[0147] X₀₆ is His or Ser,

[0148] provided that said peptide is not PTHrP(1-14).

[0149] Thus, based upon the above noted generic formula, this inventionprovides biologically active compounds of SEQ ID NO: 1 and derivativesthereof. In one specific embodiment, this invention provides abiologically active peptide at least 85% identical to a peptideconsisting essentially of the formula:

[0150] (a) X₀₁ValSerGluX₀₂GlnLueX₀₃HisX₀₄X₅GlyLysX₆(SEQ ID NO: 1);

[0151] (b) fragments thereof containing amino acids 1-9, 1-10, 1-11,1-12, or 1-13;

[0152] (c) pharmaceutically acceptable salts thereof; or

[0153] (d) N- or C-derivatives thereof;

[0154] wherein:

[0155] X₀₁ is Ser or Ala;

[0156] X₀₂ is Ile or His;

[0157] X₀₃ is Met, Leu or Nle;

[0158] X₀₄ is Asn or Asp;

[0159] X₀₅ is Leu or Lys; and

[0160] X₀₆ is His or Ser,

[0161] provided that said peptide is not PTHrP(1-14).

[0162] As protein products, compounds of SEQ ID NO: 1 or derivativesthereof of the present invention are amenable to production by thetechnique of solution- or solid-phase peptide synthesis. The solid phasepeptide synthesis technique, in particular, has been successfullyapplied in the production of human PTH and can be used for theproduction of compounds of SEQ ID NO: 1 or derivatives thereof of thepresent invention (for guidance, see Kimura et al., supra, and seeFairwell et al., Biochem. 22:2691 (1983)). Success with producing humanPTH on a relatively large scale has been reported by Goud et al., in J.Bone Min. Res. 6(8):781 (1991), incorporated herein by reference. Thesynthetic peptide synthesis approach generally entails the use ofautomated synthesizers and appropriate resin as solid phase, to which isattached the C-terminal amino acid of the desired compounds of SEQ IDNO: 1 or derivatives thereof. Extension of the peptide in the N-terminaldirection is then achieved by successively coupling a suitably protectedform of the next desired amino acid, using either FMOC- or BOC-basedchemical protocols typically, until synthesis is complete. Protectinggroups are then cleaved from the peptide, usually simultaneously withcleavage of peptide from the resin, and the peptide is then isolated andpurified using conventional techniques, such as by reversed phase HPLCusing acetonitrile as solvent and trifluoroacetic acid as ion-pairingagent. Such procedures are generally described in numerous publicationsand reference may be made, for example, to Stewart and Young, “SolidPhase Peptide Synthesis,” 2nd Edition, Pierce Chemical Company,Rockford, Ill. (1984). It will be appreciated that the peptide synthesisapproach is required for production of SEQ ID NO: 1 and derivativesthereof variants which incorporate amino acids that are not geneticallyencoded.

[0163] In a further aspect of the invention, any amino-acidsubstitutions at positions 1-9, and more particularly those amino acidsubstitutions at amino acid positions 10, 11, 12, 13, and/or 14, whichdo not destroy the biological activity of the PTH/PTHrP peptide analogto antagonize or agonize the PTH-1/PTH-2 receptor (as determined byassays known to the skilled artisan and discussed below), are alsoincluded within the scope of the present invention.

[0164] The synthetic analog of bovine PTH, PTH(3-34) has been recognizedas a potent PTH antagonist in vitro. Variants of PTH lacking N-terminalamino acids 1-2 and 1-7, were shown to be devoid of agonist activity andcapable of antagonist activity (Born, W. et al., Endocrinol.23:1848-1853 (1988)). Preferred potential antagonist variants of SEQ IDNO: 1 of this invention are variants truncated at the N-terminus.

[0165] When a variant is truncated by one amino acid at the N-terminus,it is termed PTH or PTHrP(2-14), in that it lacks amino acid residue #1but contains amino acid residues #2-14. When a variant is truncated byone amino acid at the C-terminus, it is termed PTH or PTHrP(1-13), inthat it lacks amino acid residue #14 but contains amino acid residues#1-13.

[0166] In accordance with another aspect of the present invention,substituents may be attached to the free amine of the N-terminal aminoacid of compounds of SEQ ID NO: 1 or derivatives thereof by standardmethods known in the art. For example, alkyl groups, e.g., C₁₋₁₂alkyl,may be attached using reductive alkylation. Hydroxyalkyl groups, e.g.C₁₋₁₂hydroxyalkyl, may also be attached using reductive alkylationwherein the free hydroxy group is protected with a t-butyl ester. Acylgroups, e.g., COE₁, may be attached by coupling the free acid, e.g.,E₁COOH, to the free amino of the N-terminal amino acid. Alsocontemplated within the scope of this invention are those compounds ofSEQ ID NO:1 and derivatives thereof that alter secondary or tertiarystructure, or stability of compounds of SEQ ID NO: 1 or derivativesthereof which still retain biological activity. Such derivatives mightbe achieved through lactam cyclization, disulfide bonds, or other meansknown to a person of ordinary skill in the art.

[0167] Among the preferred embodiments are those compounds which mayserve as agonists of the PTH-1/PTH-2 receptor. In particular, preferredembodiments are those compounds where X₀₁ is Ala; X₀₂ is Ile; X₀₃ isMet; X₀₄ is Asn; X₀₅ is Leu; and X₀₆ is His. The amino acid sequence ofthis preferred embodiment is thusAlaValSerGluIleGlnLeuMetHisAsnLeuGlyLysHis (SEQ ID NO: 5) or derivativesthereof.

[0168] Another set of the preferred embodiments are those compoundshaving a five amino acid deletion at the carboxy terminus of SEQ ID NO:1 where X₀₁ is Ala; X₀₂ is Ile; and X₀₃ is Met. The amino acid sequenceof this preferred embodiment is thus AlaValSerGluIleGlInLeuMetHis (SEQID NO: 6) or derivatives thereof.

[0169] Another set of preferred embodiments are those compounds whereX₀₁ is Ala; X₀₂ is Ile; X₀₃ is Leu; X₀₄ is Asp; X₀₅ is Lys; and X₀₆ isSer. The amino acid sequence of this preferred embodiment is thusAlaValSerGluIleGlnLeuLeuHisAsp LysGlyLysSer (SEQ ID NO: 2) orderivatives thereof.

[0170] Another set of preferred embodiments are those compounds having afive amino acid deletion at the carboxy terminus of SEQ ID NO: 1 whereX₀₁ is Ala; X₀₂ is Ile; and X₀₃ is Leu. The amino acid sequence of thispreferred embodiment is thus AlaValSerGluIleGlnLeuLeuHis (SEQ ID NO: 7)or derivatives thereof.

[0171] Another set of preferred embodiments are those compounds having afive amino acid deletion at the carboxy terminus of SEQ ID NO: 1 whereX₀₁ is Ala; X₀₂ is His; and X₀₃ is Leu. The amino acid sequence of thispreferred embodiment is thus AlaValSerGluHisGlnLeuLeuHis (SEQ ID NO: 8)or derivatives thereof.

[0172] Another set of the preferred embodiments are those compoundswhere X₀ is Ser; X₀₂ is His; X₀₃ is Leu; X₀₄ is Asp; X₀₅ is Lys; and X₀₆is Ser. The amino acid sequence of this preferred embodiment is thusSerValSerGluHisGlnLeu LeuHisAspLysGlyLysSer (SEQ ID NO: 9) orderivatives thereof.

[0173] Another set of the preferred embodiments are those compoundshaving a five amino acid deletion at the carboxy terminus of SEQ ID NO:1 where X₀ l is Ser; X₀₂ is His; and X₀₃ is Leu. The amino acid sequenceof this preferred embodiment is thus SerValSerGluHisGlnLeuLeuHis (SEQ IDNO: 10) or derivatives thereof.

[0174] Among the preferred embodiments are those compounds which mayserve as antagonists of the PTH-1/PTH-2 receptor. In particular,preferred embodiments are those compounds having a single amino aciddeletion at the amino terminus of SEQ ID NO: 1 where X₀₂ is Ile; X₀₃ isMet; X₀₄ is Asn; X₀₅ is Leu; and X₀₆ is His. The amino acid sequence ofthis preferred embodiment is thusValSerGlulIeGlnLeuMetHisAsnLeuGlyLysHis (SEQ ID NO: 11) or derivativesthereof.

[0175] Yet another set of preferred antagonist embodiments are thosecompounds having a single amino acid deletion at the amino terminus ofSEQ ID NO: 1 where X₀₂ is Ile; X₀₃ is Leu; X₀₄ is Asp; X₀₅ is Lys; andX₀₆ is Ser. The amino acid sequence of this preferred embodiment is thusValSerGluIleGlnLeuLeuHisAspLys GlyLysSer (SEQ ID NO: 12) or derivativesthereof.

[0176] Yet another set of preferred antagonist embodiments are thosecompounds having a single amino acid deletion at the amino terminus ofSEQ ID NO: 1 where X₀₂ is His; X₀₃ is Leu; X₀₄ is Asp; X₀₅ is Lys; andX₀₆ is Ser. The amino acid sequence of this preferred embodiment is thusValSerGluHisGlnLeuLeuHisAsp LysGlyLysSer (SEQ ID NO: 13) or derivativesthereof.

[0177] II. Biological Characterization of Compounds of SEQ ID NO:1 andDerivatives Thereof

[0178] Functional characterization of the biological properties of thecompounds of SEQ ID NO:1 and derivatives thereof was performed bybioassays that measure ligand-stimulated cAMP accumulation.

[0179] A. Stimulation of Cyclic AMP Accumulation by Compounds of SEQ IDNO: 1 or Derivatives Thereof

[0180] Intracellular cAMP accumulation was measured as describedpreviously (Abou-Samra et al., J. Biol. Chem. 262:1129, 1986). Cells in24-well plates were rinsed with culture medium containing 0.1% BSA and 2mM IBMX. The cells were then incubated with compounds of SEQ ID NO: 1 orderivatives thereof for 60 min. at 21° C. The supernatant was removedand the cells immediately frozen by placing the whole plate in dry icepowder. Intracellular cAMP was extracted by thawing the cells in 1 ml of50 mM HCl and analyzed by a specific radioimmunoassay using an anti-cAMPantibody (e.g., Sigma, St. Louis, Mo.). A cAMP analog(2′-O-monosuccinyl-adenosine3′:5′-cyclic monophosphate tyrosyl methylester, obtained from Sigma) which was used a tracer for cAMP wasiodinated by the chloramine T method. Free iodine was removed byadsorbing the iodinated cAMP analog onto a C18 Sep-pak cartridge(Waters, Milford, Mass.). After washing with dH₂O, the iodinated cAMPanalog was eluted from the Sep-pak Cartridge with 40% acetonitrille(ACN) and 0.1% trifluoroacetic acid (TFA). The iodinated cAMP analog waslyophilized, reconstituted in 1 ml 0.1% TFA, and injected into a C18reverse phase HPLC column (Waters). The column was equilibrated with 10%ACN in 0.1% TFA, and eluted with gradient of 10-30% ACN in 0.1% TFA.This allows separation of the mono-iodinated cAMP analog from thenon-iodinated cAMP analog. The tracer is stable for up to 4 months whenstored at −20° C. The standard used for the assay, adenosine3′:5′-cyclic monophosphate, was purchased from Sigma. Samples (1-10 82 Iof HCl extracts) or standards (0.04-100 fmol/tube) were diluted in 50 mMNa-acetate (pH 5.5), and acetylated with 10 μl of mixture oftriethylamine and acetic anhydride (2:1 vol:vol). After acetylation,cAMP antiserum (100 μl) was added from a stock solution (1:4000) made inPBS (pH 7.4), 5 mM EDTA and 1% normal rabbit serum. The tracer wasdiluted in PBS (pH 7.4) with 0.1% BSA, and added (20,000 cpm/tube). Theassay was incubated at 4° C. overnight. The bound tracer wasprecipitated by adding 100 μl of goat anti-rabbit antiserum (1:20 inPBS) and 1 ml of 7% polyethyleneglycol (MW 5000-6000), centrifuging at2000 rpm for 30 min. at 4° C. The supernatant was removed and the boundradioactivity was counted in a gamma-counter (Micromedic). To computethe cAMP data, logit calculations were performed in Excel spreadsheets.Typically, the assay sensitivity is 0.1 fmol/tube, and the standardconcentration that displaces 50% of tracer is 5 fmol/tube.

[0181] B. Binding of Compounds of SEQ ID NO: 1 or Derivatives Thereof toCloned Receptors Expressed on COS Cells

[0182] In addition to the cAMP accumulation assay described below,compounds of SEQ ID NO: 1 or derivatives thereof may also be iodinatedand used in a radioreceptor-based assay in transiently transfected COScells. COS-7 cells are grown in 15 cm plates in DMEM, 10%heat-inactivated FBS, 10 mg/L gentamycin until 80-90% confluent.Twenty-four hours after transfection by the DEAE/Dextran method(Sambrook et al., supra), with 1-2 μg of plasmid DNA, the cells aretrypsinized and replated in multiwell plastic dishes (16 or 35 mmdiameter, Costar, Cambridge, Mass.) at a cell concentration of 5×10⁴cells/cm². Cell number increased only slightly after transfection. Aftercontinuing culture for another 48 h, radioreceptor assays are performed.The culture medium is replaced with buffer containing 50 mM Tris-HCL (pH7.7), 100 mM NaCl, 2 mM CaCl₂, 5 mM KCL, 0.5% heat-inactivated fetalbovine serum (GIBCO), and 5% heat-inactivated horse serum (KC BiologicalInc., Lenexa, Kans.) immediately before studies are initiated. Unlessotherwise indicated, studies are conducted with cells incubated in thisbuffer at 15° C. for 4 h with 4×10⁵ cpm/ml (9.6×10⁻¹¹ M) of ¹²⁵I-labeled[Ala¹]PTH(1-14) amide or ¹²⁵I-labeled [Nle⁸]PTH(1-14).

[0183] III. Vectors, Host Cells, and Recombinant Expression

[0184] The present invention also relates to vectors that comprise apolynucleotide of the present invention, and host cells which aregenetically engineered with vectors of the invention and the productionof polypeptides of the invention by recombinant techniques. Cell-freetranslation systems can also be employed to produce such proteins usingRNAs derived from the DNA constructs of present invention.

[0185] For recombinant production, host cells can be geneticallyengineered to incorporate expression systems or portions thereof forpolynucleotides of the present invention. Introduction ofpolynucleotides into host cells can be effected by methods described inmany standard laboratory manuals, such as Davis et al., Basic Methods inMolecular Biology (1986) and Sambrook et al., Molecular Cloning: ALaboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. (1989) such as calcium phosphate transfection,DEAE-dextran mediated transfection, transvection, microinjection,cationic lipid-mediated transfection, electroporation, transduction,scrape loading, ballistic introduction or infection.

[0186] Representative examples of appropriate hosts include bacterialcells, such as streptococci, staphylococci, E. coli, Streptomyces andBacillus subtilis cells; fungal cells, such as yeast cells andAspergillus cells; insect cells such as Drosophila S2 and Spodoptera Sf9cells; animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, 293 andBowes melanoma cells; and plant cells.

[0187] A great variety of expression systems can be used. Such systemsinclude, among others, chromosomal, episomal and virus-derived systems,e.g., vectors derived from bacterial plasmids, from bacteriophage, fromtransposons, from yeast episomes, from insertion elements, from yeastchromosomal elements, from viruses such as baculoviruses, papovaviruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses,pseudorabies viruses, and retroviruses, and vectors derived fromcombinations thereof, such as those derived from plasmid andbacteriophage genetic elements, such as cosmids and phagemids. Theexpression systems may contain control regions that regulate as well asengender expression. Generally, any system or vector suitable tomaintain, propagate or express polynucleotides to produce a polypeptidein a host may be used. The appropriate nucleotide sequence may beinserted into an expression system by any of a variety of well-known androutine techniques, such as, for example, those set forth in Sambrook etal., Molecular Cloning: A Laboratory Manual (supra).

[0188] RNA vectors may also be utilized for the expression of thenucleic acids encoding compounds of SEQ ID NO: 1 or derivatives thereofdisclosed in this invention. These vectors are based on positive ornegative strand RNA viruses that naturally replicate in a wide varietyof eukaryotic cells (Bredenbeek, P. J. & Rice, C. M., Virology 3:297-310, 1992). Unlike retroviruses, these viruses lack an intermediateDNA life-cycle phase, existing entirely in RNA form. For example, alphaviruses are used as expression vectors for foreign proteins because theycan be utilized in a broad range of host cells and provide a high levelof expression; examples of viruses of this type include the Sindbisvirus and Semliki Forest virus (Schlesinger, S., TIBTECH 11:18-22, 1993;Frolov, I., et al., Proc. Natl. Acad. Sci. (USA) 93: 11371-11377, 1996).As exemplified by Invitrogen's Sinbis expression system, theinvestigator may conveniently maintain the recombinant molecule in DNAform (pSinrep5 plasmid) in the laboratory, but propagation in RNA formis feasible as well. In the host cell used for expression, the vectorcontaining the gene of interest exists completely in RNA form and may becontinuously propagated in that state if desired.

[0189] For secretion of the translated protein into the lumen of theendoplasmic reticulum, into the periplasmic space or into theextracellular environment appropriate secretion signals may beincorporated into the desired polypeptide. These signals may beendogenous to the polypeptide or they may be heterologous signals.

[0190] The expression of a DNA sequence requires that the DNA sequencebe “operably linked” to DNA sequences which contain transcriptional andtranslational regulatory information. An operable linkage is a linkagein which the control or regulatory DNA sequences and the DNA sequencesought to be expressed are connected in such a way as to permit geneexpression. The precise nature of the “control regions” needed for geneexpression may vary from organism to organism, but shall in generalinclude a promoter region which, in prokaryotic cells, contains both thepromoter (which directs the initiation of RNA transcription) as well asDNA sequences which, when transcribed into RNA, will signal theinitiation of protein synthesis. Regulatory regions in eukaryotic cellswill in general include a promoter region sufficient to direct theinitiation of RNA synthesis.

[0191] Two DNA sequences are said to be operably linked if the nature ofthe linkage between the two DNA sequences does not (1) result in theintroduction of a frameshift mutation, (2) interfere with the ability ofthe promoter region sequence to direct the transcription of the fusionprotein-encoding sequence or (3) interfere with the ability of thefusion protein-encoding sequence to be transcribed by the promoterregion sequence. Thus, a promoter region would be operably linked to aDNA sequence if the promoter were capable of transcribing that DNAsequence.

[0192] The joining of various DNA fragments, to produce the expressionvectors of this invention is performed in accordance with conventionaltechniques, employing blunt-ended or staggered-ended termini forligation, restriction enzyme digestion to provide appropriate termini,filling in of cohesive ends as appropriate, alkali and phosphatasetreatment to avoid undesirable joining, and ligation with appropriateligates. In the case of a fusion protein, the genetic construct encodesan inducible promoter which is operably linked to the 5′ gene sequenceof the fusion protein to allow efficient expression of the fusionprotein.

[0193] To express compounds of SEQ ID NO: 1 or a derivative thereof in aprokaryotic cell (such as, for example, E. coli, B. subtilis,Pseudomonas, Streptomyces, etc.), it is necessary to operably link theSEQ ID NO: 1-encoding DNA sequence to a functional prokaryotic promoter.Such promoters may be either constitutive or, more preferably,regulatable (i.e., inducible or derepressible). Examples of constitutivepromoters include the int promoter of bacteriophage λ, the bla promoterof the β-lactamase gene of pBR322, and the CAT promoter of thechloramphenicol acetyl transferase gene of pBR325, etc. Examples ofinducible prokaryotic promoters include the major right and leftpromoters of bacteriophage λ, (PL and PR), the trp, recA. lacZ. lacI.and gal promoters of E. coli, the α-amylase (Ulmanen, I. et al., J.Bacteriol. 162:176-182 (1985)), and the σ-28-specific promoters of B.subtilis (Gilman, M. Z. et al., Gene 32:11-20 (1984)), the promoters ofthe bacteriophages of Bacillius (Gryczan, T. J., In: The MolecularBiology of the Bacilli, Academic Press, Inc., NY (1982)), andStreptomyces promoters (Ward, J. M. et al., Mol. Gen. Genet. 203:468-478(1986)). Prokaryotic promoters are reviewed by Glick, B. R., J. Ind.Microbiol. 1:277-282 (1987); Cenatiempo, Y., Biochimie 68:505-516(1986)); and Gottesman, S., Ann. Rev. Genet. 18:415-442 (1984)).

[0194] The preferred prokaryotic promoter for this invention is the E.coli trp promoter, which is inducible with indole acrylic acid.

[0195] If expression is desired in a eukaryotic cell, such as yeast,fungi, mammalian cells, or plant cells, then it is necessary to employ apromoter capable of directing transcription in such a eukaryotic host.Preferred eukaryotic promoters include the promoter of the mousemetallothionein I gene (Hamer, D. et al., J. Mol. Appl. Gen. 1:273-288(1982)); the TK promoter of Herpes virus (McKnight, S., Cell 31:355-365(1982)); the SV40 early promoter (Benoist, C., et al., Nature (London)290:304-310 (1981)); and the yeast gal4 gene promoter (Johnston, S. A.,et al., Proc. Natl. Acad. Sci. (USA) 79:6971-6975 (1982); Silver, P. A.,et al., Proc. Natl. Acad. Sci. (USA) 81:5951-5955 (1984)).

[0196] Preferably, the introduced gene sequence will be incorporatedinto a plasmid or viral vector capable of autonomous replication in therecipient host. Any of a wide variety of vectors may employed for thispurpose. Factors of importance in selecting a particular plasmid orviral vector include: the ease with which recipient cells that containthe vector may be recognized and selected from those recipient cellswhich do not contain the vector; the number of copies of the vectorwhich are desired in a particular host; and whether it is desirable tobe able to “shuttle” the vector between host cells of different species.

[0197] Preferred prokaryotic vectors include plasmids such as thosecapable of replication in E. coli (such as, for example, pBR322, ColE1,pSC101, pACYC 184, πVX. Such plasmids are, for example, disclosed byManiatis, T., et al., In: Molecular Cloning, A Laboratory Manual, ColdSpring Harbor Press, Cold Spring Harbor, N.Y. (1982)). Preferred plasmidexpression vectors include the pGFP-1 plasmid described in Gardella etal., J. Biol. Chem 265:15854-15859 (1989), or a modified plasmid basedupon one of the pET vectors described by Studier and Dunn, Methods inEnzymology 185: 60-89 (1990). Bacillus plasmids include pC194, pC221,pT127, etc. Such plasmids are disclosed by Gryczan, T. In: The MolecularBiology of the Bacilli, Academic Press, NY pp. 307-329 (1982). SuitableStreptomyces plasmids include pIJIOI (Kendall, K. J. et al., J.Bacteriol. 169:4177-4183 (1987)), and streptomyces bacteriophages suchas φC31 (Chater, K. F. et al., In: Sixth International Symposium onActinomycetales Biology, Akademiai Kaido, Budapest, Hungary, pp.45-54(1986)). Pseudomonas plasmids are reviewed by John, J. F. et al., Rev.Infect. Dis. 8:693-704 (1986)), and Izaki, K., Jon. J. Bacteriol.33:729-742 (1978)).

[0198] Preferred eukaryotic expression vectors include, withoutlimitation, BPV, vaccinia, 2-micron circle etc. Such expression vectorsare well known in the art (Botstein, D., et al., Miami Wntr. Symp.19:265-274 (1982); Broach, J. R., In: The Molecular Biology of the YeastSaccharomyces: Life Cycle and Inheritance, Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y. pp. 445-470 (1981); Broach, J. R.,Cell 28:203-204 (1982); Bollon, D. P., et al., J. Clin. Hematol. Oncol.10:39-48 (1980); Maniatis, T., In: Cell Biology. A ComprehensiveTreatise, Vol. 3, Gene Expression, Academic Press, NY, pp. 563-608(1980)).

[0199] In addition to microorganisms, cultures of cells derived frommulticellular organisms may also be used as hosts. In principle, anysuch cell culture is workable, whether from vertebrate or invertebratecellular sources. Interest, however, has been greater with cells fromvertebrate sources. Examples of useful vertebrate host cell lines areVERO and HeLa cells, Chinese hamster ovary (CHO) cell lines, WI138, BHK,COS-7, and MDCK cell lines. Expression vectors for such cells ordinarilyinclude (if necessary) an origin of replication, a promoter located infront of or upstream to the gene to be expressed, along with anynecessary ribosome binding sites, RNA splice sites, polyadenylationsite, and transcriptional terminator sequences.

[0200] For use in mammalian cells, the control functions on theexpression vectors are often provided by viral material. For example,commonly used promoters are derived from polyoma, Adenovirus 2, SimianVirus 40 (SV40) and cytomegalovirus. The early and late promoters ofSV40 virus are particularly useful because both are obtained easily fromthe virus as a fragment which also contains the SV40 vial origin ofreplication (Fiers et al., Nature 273:113 (1978)).

[0201] An origin of replication may be provided either by constructionof the vector to include an exogenous origin, such as may be derivedfrom SV40 or other viral (e.g. Polyoma, Adeno, VSV, BPV) source or maybe provided by the host cell chromosomal replication mechanism. If thevector is integrated into the host cell chromosome, the latter is oftensufficient.

[0202] If cells without formidable cell membrane barriers are used ashost cells, transfection is carried out by the calcium phosphateprecipitation method as described by Graham and Van der Erb, Virology52:546 (1978). However, other methods for introducing DNA into cells,such as by nuclear injection or by protoplast fusion may also be used.In the case of gene therapy, the direct naked plasmid or viral DNAinjection method, with or without transfection-facilitating agents suchas, without limitation, liposomes, provides an alternative approach tothe current methods of in vivo or in vitro transfection of mammaliancells. If prokaryotic cells or cells which contain substantial cell wallconstructions are used, the preferred method of transfection is calciumtreatment, using calcium chloride as described in Cohen et al., Proc.Natl. Acad. Sci. USA 69:2110 (1972).

[0203] IV. Utility and Administration of Compounds of SEQ ID NO:1 orDerivatives Thereof

[0204] Compounds of SEQ ID NO: 1 or derivatives thereof of thisinvention are useful for the prevention and treatment of a variety ofmammalian conditions manifested by loss of bone mass. In particular, thecompounds of this invention are indicated for the prophylaxis andtherapeutic treatment of osteoporosis and osteopenia in humans.Furthermore, the compounds of this invention are indicated for theprophylaxis and therapeutic treatment of other bone diseases. Thecompounds of this invention are indicated for the prophylaxis andtherapeutic treatment of hypoparathyroidism. Finally, the compounds ofthis invention are indicated for use as agonists for fracture repair andas antagonists for hypercalcemia.

[0205] In general, compounds of SEQ ID NO: 1 or derivatives thereof ofthis invention, or salts thereof, are administered in amounts betweenabout 0.01 and 1 μg/kg body weight per day, preferably from about 0.07to about 0.2 μg/kg body weight per day. For a 50 kg human femalesubject, the daily dose of biologically active compounds of SEQ ID NO: 1or derivatives thereof is from about 0.5 to about 50 μgs, preferablyfrom about 3.5 to about 10 μgs. In other mammals, such as horses, dogs,and cattle, higher doses may be required. This dosage may be deliveredin a conventional pharmaceutical composition by a single administration,by multiple applications, or via controlled release, as needed toachieve the most effective results, preferably one or more times dailyby injection. For example, this dosage may be delivered in aconventional pharmaceutical composition by nasal insufflation.

[0206] The selection of the exact dose and composition and the mostappropriate delivery regimen will be influenced by, inter alia, thepharmacological properties of the selected compounds of SEQ ID NO: 1 orderivatives thereof, the nature and severity of the condition beingtreated, and the physical condition and mental acuity of the recipient.

[0207] Representative preferred delivery regimens include, withoutlimitation, oral, parenteral (including subcutaneous, transcutaneous,intramuscular and intravenous), rectal, buccal (including sublingual),transdermal, and intranasal insufflation.

[0208] Pharmaceutically acceptable salts retain the desired biologicalactivity of the compounds of SEQ ID NO: 1 or derivatives thereof withouttoxic side effects. Examples of such salts are (a) acid addition saltsformed with inorganic acids, for example hydrochloric acid, hydrobromicacid, sulfuric acid, phosphoric acid, nitric acid and the like; andsalts formed with organic acids such as, for example, acetic acid,oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid,gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid,tannic acid, pamoic acid, alginic acid, polyglutamic acid,naphthalenesulfonic acids, naphthalene disulfonic acids,polygalacturonic acid and the like; (b) base addition salts formed withpolyvalent metal cations such as zinc, calcium, bismuth, barium,magnesium, aluminum, copper, cobalt, nickel, cadmium, and the like; orwith an organic cation formed from N,N′-dibenzylethylenediamine orethylenediamine; or (c) combinations of (a) and (b), e.g., a zinctannate salt and the like.

[0209] A further aspect of the present invention relates topharmaceutical compositions comprising as an active ingredient compoundsof SEQ ID NO: 1 or derivatives thereof of the present invention, orpharmaceutically acceptable salt thereof, in admixture with apharmaceutically acceptable, non-toxic carrier. As mentioned above, suchcompositions may be prepared for parenteral (subcutaneous,transcutaneous, intramuscular or intravenous) administration,particularly in the form of liquid solutions or suspensions; for oral orbuccal administration, particularly in the form of tablets or capsules;for rectal, transdermal administration; and for intranasaladministration, particularly in the form of powders, nasal drops oraerosols.

[0210] The compositions may conveniently be administered in unit dosageform and may be prepared by any of the methods well-known in thepharmaceutical art, for example as described in Remington'sPharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa.,(1985), incorporated herein by reference. Formulations for parenteraladministration may contain as excipients sterile water or saline,alkylene glycols such as propylene glycol, polyalkylene glycols such aspolyethylene glycol, oils of vegetable origin, hydrogenated naphthalenesand the like. For oral administration, the formulation can be enhancedby the addition of bile salts or acylcarnitines. Formulations for nasaladministration may be solid and may contain excipients, for example,lactose or dextran, or may be aqueous or oily solutions for use in theform of nasal drops or metered spray. For buccal administration typicalexcipients include sugars, calcium stearate, magnesium stearate,pregelatinated starch, and the like.

[0211] When formulated for the most preferred route of administration,nasal administration, the absorption across the nasal mucous membranemay be enhanced by surfactant acids, such as for example, glycocholicacid, cholic acid, taurocholic acid, ethocholic acid, deoxycholic acid,chenodeoxycholic acid, dehydrocholic acid, glycodeoxycholic acid,cyclodextrins and the like in an amount in the range between about 0.2and 15 weight percent, preferably between about 0.5 and 4 weightpercent, most preferably about 2 weight percent.

[0212] Delivery of the compounds of the present invention to the subjectover prolonged periods of time, for example, for periods of one week toone year, may be accomplished by a single administration of a controlledrelease system containing sufficient active ingredient for the desiredrelease period. Various controlled release systems, such as monolithicor reservoir-type microcapsules, depot implants, osmotic pumps,vesicles, micelles, liposomes, transdermal patches, iontophoreticdevices and alternative injectable dosage forms may be utilized for thispurpose. Localization at the site to which delivery of the activeingredient is desired is an additional feature of some controlledrelease devices, which may prove beneficial in the treatment of certaindisorders.

[0213] One form of controlled release formulation contains thepolypeptide or its salt dispersed or encapsulated in a slowly degrading,non-toxic, non-antigenic polymer such as copoly(lactic/glycolic) acid,as described in the pioneering work of Kent, Lewis, Sanders, and Tice,U.S. Pat. No. 4,675,189, incorporated by reference herein. The compoundsor, preferably, their relatively insoluble salts, may also be formulatedin cholesterol or other lipid matrix pellets, or silastomer matriximplants. Additional slow release, depot implant or injectableformulations will be apparent to the skilled artisan. See, for example,Sustained and Controlled Release Drug Delivery Systems, J. R. Robinsoned., Marcel Dekker, Inc., New York, 1978, and R. W. Baker, ControlledRelease of Biologically Active Agents, John Wiley & Sons, New York,1987, incorporated by reference herein.

[0214] Like PTH, the PTH variants may be administered in combinationwith other agents useful in treating a given clinical condition. Whentreating osteoporosis and other bone-related disorders for example, thePTH variants may be administered in conjunction with a dietary calciumsupplement or with a vitamin D analog (see U.S. Pat. No. 4,698,328).Alternatively, the PTH variant may be administered, preferably using acyclic therapeutic regimen, in combination with bisphosphonates, asdescribed for example in U.S. Pat. No. 4,761,406, or in combination withone or more bone therapeutic agents such as, without limitation,calcitonin and estrogen.

[0215] V. Receptor-Signaling Activities of Compounds of SEQ ID NO: 1 orDerivatives Thereof

[0216] A crucial step in the expression of hormonal action is theinteraction of hormones with receptors on the plasma membrane surface oftarget cells. The formation of hormone-receptor complexes allows thetransduction of extracellular signals into the cell to elicit a varietyof biological responses.

[0217] A. Screening for PTH-1 Receptor Antagonists and Agonists

[0218] Polypeptides of the invention may be screened for their agonisticor antagonistic properties using the cAMP accumulation assay. Cellsexpressing PTH-1 receptor on the cell surface are incubated with nativePTH(1-84) for 5-60 minutes at 37° C., in the presence of 2 mM IBMX(3-isobutyl-1-methyl-xanthine, Sigma, St. Louis, Mo.). Cyclic AMPaccumulation is measured by specific radio-immunoassay, as describedabove. A compound of SEQ ID NO: 1 or a derivative thereof that competeswith native PTH(1-84) for binding to the PTH-1 receptor, and thatinhibits the effect of native PTH(1-84) on cAMP accumulation, isconsidered a competitive antagonist. Such a compound would be useful fortreating hypercalcemia.

[0219] Conversely, a compound of SEQ ID NO: 1 or a derivative thereofthat does not compete with native PTH(1-84) for binding to the PTH-1receptor, but which still prevents native PTH(1-84) activation of cAMPaccumulation (presumably by blocking the receptor activation site) isconsidered a non-competitive antagonist. Such a compound would be usefulfor treating hypercalcemia.

[0220] A compound of SEQ ID NO: 1 or a derivative thereof that competeswith native PTH(1-84) for binding to the PTH-1 receptor, and whichstimulates cAMP accumulation in the presence or absence of nativePTH(1-84) is a competitive agonist. A compound of SEQ ID NO: 1 or aderivative thereof that does not compete with native PTH(1-84) forbinding to the PTH-1 receptor but which is still capable of stimulatingcAMP accumulation in the presence or absence of native PTH(1-84), orwhich stimulates a higher cAMP accumulation than that observed by acompound of SEQ ID NO: 1 or a derivative thereof alone, would beconsidered a non-competitive agonist.

[0221] VI. Therapeutic Uses of Compounds of SEQ ID NO: 1 or DerivativesThereof

[0222] Some forms of hypercalcemia and hypocalcemia are related to theinteraction between PTH and PTHrP and the PTH-1 and PTH-2 receptors.Hypercalcemia is a condition in which there is an abnormal elevation inserum calcium level; it is often associated with other diseases,including hyperparathyroidism, osteoporosis, carcinomas of the breast,lung and prostate, epidermoid cancers of the head and neck and of theesophagus, multiple myeloma, and hypernephroma. Hypocalcemia, acondition in which the serum calcium level is abnormally low, may resultfrom a deficiency of effective PTH, e.g., following thyroid surgery.

[0223] Nucleic acids of the invention which encode compounds of SEQ IDNO: 1 or derivatives thereof may also be linked to a selectedtissue-specific promoter and/or enhancer and the resultant hybrid geneintroduced, by standard methods (e.g., as described by Leder et al.,U.S. Pat. No. 4,736,866, herein incorporated by reference), into ananimal embryo at an early developmental stage (e.g., the fertilizedoocyte stage), to produce a transgenic animal which expresses elevatedlevels of compounds of SEQ ID NO: 1 or derivatives thereof in selectedtissues (e.g., the osteocalcin promoter for bone). Such promoters areused to direct tissue-specific expression of compounds of SEQ ID NO: 1or derivatives thereof in the transgenic animal.

[0224] In addition, any other amino-acid substitutions of a nature,which do not destroy the ability of the PTH/PTHrP analog to antagonizeor agonize the PTH-1/PTH-2 receptor (as determined by assays known tothe skilled artisan and discussed below), are included in the scope ofthe present invention.

[0225] By “agonist” is intended a ligand capable of enhancing orpotentiating a cellular response mediated by the PTH-1 receptor. By“antagonist” is intended a ligand capable of inhibiting a cellularresponse mediated by the PTH-1 receptor. Whether any candidate “agonist”or “antagonist” of the present invention can enhance or inhibit such acellular response can be determined using art-known proteinligand/receptor cellular response or binding assays, including thosedescribed elsewhere in this application.

[0226] In accordance with yet a further aspect of the invention, thereis provided a method for treating a medical disorder that results fromaltered or excessive action of the PTH-1 receptor, comprisingadministering to a patient a therapeutically effective amount of acompound of SEQ ID NO: 1 or a derivative thereof sufficient to inhibitactivation of the PTH-1 receptor of said patient.

[0227] In this embodiment, a patient who is suspected of having adisorder resulting from altered action of the PTH-1 receptor may betreated using compounds of SEQ ID NO: 1 or derivatives thereof of theinvention which are a selective antagonists of the PTH-1 receptor. Suchantagonists include compounds of SEQ ID NO: 1 or derivatives thereof ofthe invention which have been determined (by the assays describedherein) to interfere with PTH-1 receptor-mediated cell activation orother derivatives having similar properties.

[0228] To administer the antagonist, the appropriate compound of SEQ IDNO: 1 or a derivative thereof is used in the manufacture of amedicament, generally by being formulated in an appropriate carrier orexcipient such as, e. g., physiological saline, and preferablyadministered intravenously, intramuscularly, subcutaneously, orally, orintranasally, at a dosage that provides adequate inhibition of acompound of SEQ ID NO: 1 or a derivative thereof binding to the PTH-1receptor. Typical dosage would be 1 ng to 10 mg of the peptide per kgbody weight per day.

[0229] In a preferred embodiment, the compound of SEQ ID NO: 1 or aderivative thereof used in the method has a single amino acid deletionat the amino terminus. In this preferred embodiment, the PTH/PTHrPanalog is PTH(2-14)/PTHrP(2-14). In yet another preferred embodiment,the compound of SEQ ID NO: 1 or a derivative thereof used in the methodhas a two amino acid deletion at the amino terminus. In this preferredembodiment, the PTH/PTHrP analog is PTH(3-14)/PTHrP(3-14).

[0230] In accordance with yet a further aspect of the invention, thereis provided a method for treating osteoporosis, comprising administeringto a patient a therapeutically effective amount of a compound of SEQ IDNO: 1 or a derivative thereof, sufficient to activate the PTH-1 receptorof said patient. Similar dosages and administration as described abovefor the PTH/PTHrP antagonist, may be used for administration of aPTH/PTHrP agonist, e.g., for treatment of conditions such asosteoporosis, other metabolic bone disorders, and hypoparathyroidism andrelated disorders.

[0231] In a preferred embodiment, the compound of SEQ ID NO: 1 or aderivative thereof used in the method has an amino acid substitution ofalanine for serine at amino acid position 1 of compound of SEQ ID NO: 1.In this particular embodiment, the PTH derivative is [Ala¹]PTH(1-14)(SEQID NO: 5). In another preferred embodiment, the compound of SEQ ID NO: 1or a derivative thereof used in the method has an amino acidsubstitution of histidine for isoleucine at position 5 of SEQ ID NO: 1.In this particular embodiment, the PTHrP derivative is[Ile⁵]PTHrP(1-14). (SEQ ID NO: 2)

[0232] It will be appreciated to those skilled in the art that theinvention can be performed within a wide range of equivalent parametersof composition, concentration, modes of administration, and conditionswithout departing from the spirit or scope of the invention or anyembodiment thereof.

[0233] Having now fully described the invention, the same will be morereadily understood by reference to specific examples which are providedby way of illustration, and are not intended to be limiting of theinvention, unless herein specified.

EXAMPLE 1

[0234] To begin to identify the minimum length required for bioactivityof PTH and PTHrP, the inventors constructed synthetic peptides basedupon the first 14 amino acids of native human PTH. As a first steptowards optimization the inventors replaced the serine at position oneby alanine; this substitution, which corresponds to the amino acid foundat position 1 in rat and bovine PTH, as well as in all PTHrP moleculesreported so far (human, bovine, dog, rat, mouse, chicken), results in ameasurable increase in bioactivity over the background level ofbioactivity of the native PTH(1-14) peptide. The C-terminal residue ofthis new peptide, herein called [Ala¹]PTH(1-14), is amidated.

[0235] The ability of [Ala¹]PTH(1-14) to stimulate cAMP formation inCOS-7 cells expressing the cloned human PTH-1 receptor is shown inFIG. 1. A small cAMP response can be seen even with the shorter peptide[Ala¹]PTH(1-9). As expected, the carboxy-terminal fragment PTH(15-31)was inactive (FIG. 1). Each of these peptides was inactive in controlCOS-7 cells transected with a DNA vector lacking the PTH-1 receptorgene.

[0236] To demonstrate PTH(1-14) specificity, its ability to stimulatecAMP production in COS-7 cells transfected with the rat secretinreceptor (rSR)—a related Family B receptor that does not bind or respondto PTH was tested. As shown in the FIG. 6, PTH(1-14) is inactive incells expressing rSR. Thus, the response to PTH(1-14) in COS-7 cells isdependent on PTH-1 receptor expression. PTH(1-14) specificity was alsotested using the porcine renal cell line LLC-PK1, either untransfectedor stably transfected with the human PTH-1 receptor, the LLC-B7 cellline. FIG. 4 shows that the PTH(1-14) response in these cells isdependent on PTH-1 receptor expression.

EXAMPLE 2

[0237] As noted above in Example 1, even with the Ser→Ala substitution,the [Ala¹]PTH(1-14) peptide is weaker than native PTH(1-34) (FIG. 1).Thus, additional optimization of the [Ala¹]PTH(1-14) sequence to improvepotency and efficacy was pursued. As part of this optimization process,an alanine-scan of the native PTH(1-14) sequence was performed. In thisstudy, 14 different peptides were synthesized, each 14 amino acids inlength and differing from each other by having one native amino acidreplaced with alanine. This alanine-scan permits the classification ofeach individual residue in the native 1-14 sequence as either criticalfor function (intolerant) or not critical for function (tolerant). Thetolerant residues reside in one well-defined carboxy-terminal segmentthat extends from Asn-10 to His-14, whereas the intolerant residues fallwithin the Ala-1 to His-9 segment (FIG. 2). Note that Ala-1 and Ser-3were not adequately tested in this study, since position 1 is alanine inthe native rat and bovine sequences, and Ser→Ala at position 3 is aconservative substitution. For example, as predicted from thealanine-scan results, the [Ala¹]PTH(1-9) fragment, which contains all ofthe intolerant residues, exhibits some biological activity (FIG. 1).

EXAMPLE 3

[0238] The analysis extends to the PTHrP(1-14) sequence as well. Theinventors found that one monosubstitution, which replaces histidine atthe critical 5 position in PTHrP(1-14) with isoleucine. Thus, in thisparticular embodiment, the pTHrP analog is [Ile⁵]PTHrP(1-14). FIG. 7.shows the cAMP activity results obtained with [Ile5]PTHrP(1-14). LLC-B7cells were treated with the indicated peptide ligand, each at 100 μM,and then intracellular cAMP levels were measured. Substitution ofisoleucine with histidine at the critical 5 position of PTHrP results ina new analog with enhanced potency, relative to native PTHrP(1-14).

1 13 1 14 PRT Artificial Sequence UNSURE (1) Amino acid may be Ser orAla 1 Xaa Val Ser Glu Xaa Gln Leu Xaa His Xaa Xaa Gly Lys Xaa 1 5 10 214 PRT Artificial Sequence Description of Artificial Sequence syntheticpeptide 2 Ala Val Ser Glu Ile Gln Leu Leu His Asp Lys Gly Lys Ser 1 5 103 14 PRT Artificial Sequence Description of Artificial Sequencesynthetic peptide 3 Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly LysHis 1 5 10 4 14 PRT Artificial Sequence Description of ArtificialSequence synthetic peptide 4 Ala Val Ser Glu His Gln Leu Leu His Asp LysGly Lys Ser 1 5 10 5 14 PRT Artificial Sequence Description ofArtificial Sequence synthetic peptide 5 Ala Val Ser Glu Ile Gln Leu MetHis Asn Leu Gly Lys His 1 5 10 6 9 PRT Artificial Sequence Descriptionof Artificial Sequence synthetic peptide 6 Ala Val Ser Glu Ile Gln LeuMet His 1 5 7 9 PRT Artificial Sequence Description of ArtificialSequence synthetic peptide 7 Ala Val Ser Glu Ile Gln Leu Leu His 1 5 8 9PRT Artificial Sequence Description of Artificial Sequence syntheticpeptide 8 Ala Val Ser Glu His Gln Leu Leu His 1 5 9 14 PRT ArtificialSequence Description of Artificial Sequence synthetic peptide 9 Ser ValSer Glu His Gln Leu Leu His Asp Lys Gly Lys Ser 1 5 10 10 9 PRTArtificial Sequence Description of Artificial Sequence synthetic peptide10 Ser Val Ser Glu His Gln Leu Leu His 1 5 11 13 PRT Artificial SequenceDescription of Artificial Sequence synthetic peptide 11 Val Ser Glu IleGln Leu Met His Asn Leu Gly Lys His 1 5 10 12 13 PRT Artificial SequenceDescription of Artificial Sequence synthetic peptide 12 Val Ser Glu IleGln Leu Leu His Asp Lys Gly Lys Ser 1 5 10 13 13 PRT Artificial SequenceDescription of Artificial Sequence synthetic peptide 13 Val Ser Glu HisGln Leu Leu His Asp Lys Gly Lys Ser 1 5 10

What is claimed is:
 1. A biologically active peptide at least 85%identical to a peptide consisting essentially of the formula: (a)X₀₁ValSerGluX₀₂GlnLeuX₀₃HisX₀₄X₀₅GlyLysX₀₆(SEQ ID NO:1); (b) fragmentsthereof containing amino acids 1-9, 1-10, 1-11, 1-12, or 1-13; (c)pharmaceutically acceptable salts thereof; or (d) N- or C-derivativesthereof; wherein: X₀₁ is Ser or Ala; X₀₂ is Ile or His; X₀₃ is Met, Leuor Nle; X₀₄ is Asn or Asp; X₀₅ is Leu or Lys; and X₀₆ is His or Ser,provided that said peptide is not PTHrP(1-14).
 2. A biologically activepeptide consisting essentially of the formula: (a)X₀₁ValSerGluX₀₂GlnLeuX₀₃HisX₀₄X₀₅GlyLysX₀₆(SEQ ID NO:1); (b) fragmentsthereof containing amino acids 1-9, 1-10, 1-11, 1-12, or 1-13; (c)pharmaceutically acceptable salts thereof; or (d) N- or C-derivativesthereof; wherein: X₀₁ is Ser or Ala; X₀₂ is Ile or His; X₀₃ is Met, Leuor Nle; X₀₄ is Asn or Asp; X₀₅ is Leu or Lys; and X₀₆ is His or Ser,provided that said peptide is not PTHrP(1-14).
 3. The peptide of claim 1which is: AlaValSerGluIleGlnLeuMetHisAsnLeuGlyLysHis (SEQ ID NO: 5). 4.The peptide of claim 1 wherein the peptide is labeled with a labelselected from the group consisting of: radiolabel, flourescent label,bioluminescent label, or chemiluminescent label.
 5. The peptide of claim4, wherein said radiolabel is ^(99m) Tc.
 6. A pharmaceutical compositioncomprising (a) a biologically active peptide at least 85% identical to apeptide consisting essentially of the formula:SerValSerGluIleGlnLeuMetHisAsnLeuGlyLysHis (SEQ ID NO:3); (b) fragmentsthereof containing amino acids 1-9, 1-10, 1-11, 1-12, or 1-13; (c)pharmaceutically acceptable salts thereof; or (d) N- or C-derivativesthereof, and a pharmaceutically acceptable carrier.
 7. A pharmaceuticalcomposition comprising (a) a biologically active peptide consistingessentially of the formula: SerValSerGluIleGlnLeuMetHisAsnLeuGlyLysHis(SEQ ID NO :3); (b) fragments thereof containing amino acids 1-9, 1-10,1-11, 1-12, or 1-13; (c) pharmaceutically acceptable salts thereof; or(d) N- or C-derivatives thereof; and a pharmaceutically acceptablecarrier.
 8. A pharmaceutical composition comprising (a) a biologicallyactive peptide at least 85% identical to a peptide consistingessentially of the formula: AlaValSerGluHisGlnLeuLeuHisAspLysGlyLysSer(SEQ ID NO:4); (b) fragments thereof containing amino acids 1-9, 1-10,1-11, 1-12, or 1-13; (c) pharmaceutically acceptable salts thereof; or(d) N- or C-derivatives thereof; and a pharmaceutically acceptablecarrier.
 9. A pharmaceutical composition comprising (a) a biologicallyactive peptide consisting essentially of the formula:AlaValSerGluHisGlnLeuLeuHisAspLysGlyLysSer (SEQ ID NO:4); (b) fragmentsthereof containing amino acids 1-9, 1-10, 1-11, 1-12, or 1-13; (c)pharmaceutically acceptable salts thereof; or (d) N- or C-derivativesthereof; and a pharmaceutically acceptable carrier.
 10. A nucleic acidmolecule consisting essentially of a polynucleotide encoding abiologically active peptide which has an amino acid sequence selectedfrom the group consisting of: (a)X₀₁ValSerGluX₀₂GlnLeuX₀₃HisX₀₄X₀₅GlyLysX₀₆(SEQ ID NO:1); or (b)fragments thereof containing amino acids 1-9, 1-10, 1-11, 1-12, or 1-13;wherein: X₀₁ is Ser or Ala; X₀₂ is Ile or His; X₀₃ is Met, Leu or Nle;X₀₄ is Asn or Asp; X₀₅ is Leu or Lys; and X₀₆ is His or Ser, providedthat said peptide is not PTHrP(1-14).
 11. A recombinant DNA moleculecomprising: (1) an expression control region, said region in operablelinkage with (2) a polynucleotide sequence coding for a biologicallyactive peptide, wherein said peptide is selected from the groupconsisting of: (a) X₀₁ValSerGluX₀₂GlnLeuX₀₃HisX₀₄X₀₅GlyLysX₀₆(SEQ IDNO:1); or (b) fragments thereof containing amino acids 1-9, 1-10, 1-11,1-12, or 1-13; wherein: X₀₁ is Ser or Ala; X₀₂ is Ile or His; X₀₃ isMet, Leu or Nle; X₀₄ is Asn or Asp; X₀₅ is Leu or Lys; and X₀₆ is His orSer, provided that said peptide is not PTHrP(1-14).
 12. A method ofpreparing a biologically active peptide comprising introducing into ahost the recombinant DNA molecule of claim 11, and causing expression ofsaid molecule.
 13. A method for making a recombinant vector comprisinginserting a nucleic acid molecule of claim 10 into a vector.
 14. Therecombinant DNA molecule of claim 11, wherein said control regionincludes a bacterial, viral, fungal or mammalian promoter.
 15. A hostcell containing the recombinant DNA molecule of claim
 11. 16. The cellof claim 15 which is prokaryotic.
 17. The cell of claim 16 which isbacterial.
 18. The cell of claim 15 which is eukaryotic.
 19. The cell ofclaim 18 which is a yeast cell or a mammalian cell.
 20. A method fortreating mammalian conditions characterized by decreases in bone mass,which method comprises administering to a subject in need thereof aneffective bone mass-increasing amount of a biologically active peptide,wherein said peptide comprises an amino acid sequence at least 85%identical to a member selected from the group consisting essentially of:(a) X₀₁ValSerGluX₀₂GlnLeuX₀₃HisX₀₄X₀₅GlyLysX₀₆(SEQ ID NO:1); (b)fragments thereof containing amino acids 1-9, 1-10, 1-11, 1-12, or 1-13;(c) pharmaceutically acceptable salts thereof; or (d) N- orC-derivatives thereof; wherein: X₀₁ is Ser or Ala; X₀₂ is Ile or His;X₀₃ is Met, Leu or Nle; X₀₄ is Asn or Asp; X₀₅ is Leu or Lys; and X₀₆ isHis or Ser, provided that said peptide is not PTHrP(1-14); and apharmaceutically acceptable carrier.
 21. A method for treating mammalianconditions characterized by decreases in bone mass, which methodcomprises administering to a subject in need thereof an effective bonemass-increasing amount of a biologically active peptide consistingessentially of the formula: (a)X₀₁ValSerGluX₀₂GlnLeuX₀₃HisX₀₄X₀₅GlyLysX₀₆(SEQ ID NO:1); (b) fragmentsthereof containing amino acids 1-9, 1-10, 1-11, 1-12, or 1-13; (c)pharmaceutically acceptable salts thereof; or (d) N- or C-derivativesthereof wherein: X₀₁ is Ser or Ala; X₀₂ is Ile or His; X₀₃ is Met, Leuor Nle; X₀₄ is Asn or Asp; X₀₅ is Leu or Lys; and X₀₆ is His or Ser,provided that said peptide is not PTHrP(1-14); and a pharmaceuticallyacceptable carrier.
 22. A method for the treatment of a patient havingneed of a biologically active peptide comprising administering atherapeutically effective amount of a peptide, wherein said peptidecomprises an amino acid sequence at least 85% identical to a memberselected from thc group consisting essentially of: (a) a biologicallyactive peptide consisting essentially of the formula:SerValSerGluIleGlnLeuMetHisAsnLeuGlyLysHis (SEQ ID NO:3); (b) fragmentsthereof containing amino acids 1-9, 1-10, 1-11, 1-12, or 1-13; (c) N- orC-derivatives thereof; or (d) pharmaceutically acceptable salts thereof;and a pharmaceutically acceptable carrier.
 23. A method for treatingmammalian conditions characterized by decreases in bone mass, whichmethod comprises administering to a subject in need thereof an effectivebone mass-increasing amount of a biologically active peptide consistingessentially of the formula: (a) a biologically active peptide consistingessentially of the formula: SerValSerGluIleGlnLeuMetHisAsnLeuGlyLysHis(SEQ ID NO:3); (b) fragments thereof containing amino acids 1-9, 1-10,1-11, 1-12, or 1-13; (c) N- or C-derivatives thereof; or (d)pharmaceutically acceptable salts thereof; and a pharmaceuticallyacceptable carrier.
 24. A method for the treatment of a patient havingneed of a biologically active peptide comprising administering atherapeutically effective amount of a peptide, wherein said peptidecomprises an amino acid sequence at least 85% identical to a memberselected from the group consisting essentially of: (a) a biologicallyactive peptide consisting essentially of the formula:AlaValSerGluHisGlnLeuLeuHisAspLysGlyLysSer (SEQ ID NO:4); (b) fragmentsthereof containing amino acids 1-9, -10, 1-11, 1-12, or 1-13; (c) N- orC-derivatives thereof; or (d) pharmaceutically acceptable salts thereof;and a pharmaceutically acceptable carrier.
 25. A method for treatingmammalian conditions characterized by decreases in bone mass, whichmethod comprises administering to a subject in need thereof an effectivebone mass-increasing amount of a biologically active peptide consistingessentially of the formula: (a)AlaValSerGluHisGlnLeuLeuHisAspLysGlyLysSer (SEQ ID NO:4); (b) fragmentsthereof containing amino acids 1-9, 1-10, 1-11, 1-12, or 1-13; (c) N- orC-derivatives thereof; or (d) pharmaceutically acceptable salts thereof;and a pharmaceutically acceptable carrier.
 26. A method for determiningrates of bone reformation, bone resorption and/or bone remodelingcomprising administering to a patient an effective amount of a peptideof claim 4 and determining the uptake of said peptide into the bone ofsaid patient.
 27. The method of claim 20, wherein said effective bonemass-increasing amount of said peptide is administered by providing tothe patient DNA encoding said peptide and expressing said peptide invivo.
 28. The method of claim 20, wherein the condition to be treated isosteoporosis.
 29. The method of claim 20, wherein said osteoporosis isold age osteoporosis.
 30. The method of claim 20, wherein saidosteoporosis is post-menopausal osteoporosis.
 31. The method of claim20, wherein the effective amount of said peptide for increasing bonemass is from about 0.01 μg/kg/day to about 1.0 μg/kg/day.
 32. The methodof claim 20, wherein the method of administration is parenteral.
 33. Themethod of claim 20, wherein the method of administration issubcutaneous.
 34. The method of claim 20, wherein the method ofadministration is nasal insufflation.